Eukaryotic cells maintain organelles that separate many of their essential functions. These compartments contain proteins that must be specifically and efficiently targeted to the correct subcellular location. This segregation of organellar proteins needs to be maintained despite a continual movement of proteins and membranes throughout the cell. How is this achieved? Different signals in combination with sorting machinery allow cytosolic proteins to be delivered to a particular destination. Defects in the localization process have severe physiological consequences. For example, the lysosome is the primary storage site for many hydrolases. Missorting of these enzymes is implicated in a wide range of illnesses. Proper lysoscmal function is not only dependent on the presence of resident hydrolases, but also on the delivery of appropriate substrates. One of the primary pathways for macromolecular turnover and recycling in mammalian cells is autophagy. This process is induced by starvation and results in the delivery of cytoplasmic proteins and organelles to the lysosome via a double membrane vesicle. Under some conditions, this mode of uptake is very specific. The signal transduction pathway by which the nutritional conditions are sensed, the methods of achieving cargo specificity and the mechanism of vesicle formation are largely unknown. Defects in autophagy have been correlated with heart disease, cancer, neurodegenerative disorders such as Parkinson's, Huntington's and Alzheimer's diseases and susceptibility to viral and bacterial infection. The yeast vacuole is analogous to the mammalian lysosome both in terms of its cellular role and its mechanisms of protein delivery. In particular, the autophagic pathway is conserved between yeast and mammalian cells. Due to the ease of genetic approaches, yeast provides a useful model system to study this pathway. In this proposal, we will focus on the elucidation of the molecular components that direct the delivery of cytosolic proteins and organelles to the lysosome/vacuole. We will use molecular genetic and biochemical approaches to determine the signal transduction pathway that allows the nucleation of sequestering vesicles. In addition, we will reconstitute the steps of cargo packaging, vesicle formation and membrane fusion in vitro in order to understand the molecular mechanism that regulates autophagy.
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